Biochemistry 1981, 20, 5212-5221
5212
Helenius, A., McCaslin, D. R., Fries, E., & Tanford, C. (1979) Methods Enzymol. 56, 734-749. Henselman, R. A., & Cusanovich, M. A. (1974) Biochemistry 13, 5 199-5203, Hong, K., & Hubbell, W. L. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 2617-2621. Hong, K., & Hubbell, W . L. (1973) Biochemistry 12, 45 17-4523. Hubbard, R., Brown, P. K., & Bownds, M. D. (1971) Methods Enzymol. 18, 615-653. Knudson, P., & Hubbell, W. L. (1978) Membr. Biochem. 1, 297-322. Matsumoto, H., Horiuchi, K., & Yoshizawa, T. (1978) Biochim. Biophys. Acta 501, 257-268. McCaslin, D. R. (1980) Ph.D. Dissertation, Duke University. McCaslin, D. R., & Tanford, C. (1981) Biochemistry (following paper in this issue). Osborne, H. B., Sardet, C., & Helenius, A. (1974) Eur. J . Biochem. 44, 383-390. Osborne, H. B., Sardet, C., Michel-Villaz, M., & Chabre, M.
'
(1978) J. Mol. Biol. 123, 177-206. Sardet, C., Tardieu, A., & Luzzati, V. (1976) J . Mol. Biol. 105, 383-407. Small, D. M. (1971) in The Bile Acids (Nair, P. P., & Kritchevsky, D., Eds.) Vol. 1, pp 249-353, Plenum Press, New York. Stubbs, G. W., & Litman, B. J. (1978) Biochemistry 17, 220-225. Tanford, C. (1980) The Hydrophobic Effect, 2nd ed., Wiley, New York. Wald, G., & Brown, P. K. (1953) J . Gen. Physiol. 37, 189-200. Wald, C., & Brown, P. K. (1956) Nature (London) 177, 174- 17 6. Weiss, H., & Wingfield, P. (1979) Eur. J. Biochem. 99, 151-160. Yoshikami, S.,& Noll, G. N . (1978) Science (Washington, D.C.) 200, 1393-1395. Zorn, M., & Futterman, S. (1973) Arch. Biochem. Biophys. 157, 91-99.
Different States of Aggregation for Unbleached and Bleached Rhodopsin after Isolation in Two Different Detergentst Darrell R. McCaslint and Charles Tanford*
ABSTRACT:
Phospholipid-free rhodopsin has been purified in the detergents sodium cholate and octaethylene glycol n-dodecyl ether (C&& In both detergents, the native absorption spectrum of the unbleached protein is maintained; however, upon photolysis, the preparation in C&s loses its ability to recombine with 11-cis-retinal, whereas the preparation in cholate does not. The circular dichroic spectra of the protein in the two detergents are nearly identical, indicating that the secondary structure of the protein is the same in the two
detergents. The state of association of the protein in the two detergents is different. In sodium cholate, the smallest species present was found to be a trimer of the rhodopsin polypeptide chain, and this association was unaffected by exposure to light. On the other hand, in C12E8,the protein is monomeric and undergoes a nonspecific aggregation process on exposure to light. These results suggest that protein-protein interactions may play an important role in the stabilization of the native structure of rhodopsin.
L i g h t modulates several enzymatic activities found in rod outer segments which suggests that these activities may play a role in the visual process [see Hubbell & Bownds (1979) for a brief review]. Rhodopsin is the only component of rod outer segments capable of direct interaction with visible light, and therefore, the modulated enzymatic activities must be either directly or indirectly coupled to light absorption by rhodopsin. For a complete understanding of the interactions involved in these processes, it is necessary to isolate each component in a chemically well-defined state. Since rhodopsin and possibly other components are membrane bound, detergents are required in the isolation procedures. Detergent-
solubilized preparations are amenable to the study of interactions by classical techniques of physical biochemistry, and furthermore, they provide a vehicle by which the various components can be reconstituted into the more nearly native milieu of a phospholipid vesicle. It is imperative, however, that the native properties of all components be preserved in the presence of the detergent in which one is studying the interactions or that any alteration in a native property be reversible or demonstrably not involved in the particular interaction under study. Since rhodopsin must play a centeral role in any light-modulated response, it is essential that a detergent system be found in which the properties of membrane-bound rhodoDsin are fullv Dreserved. Many d e t e r g e k can solubilize rhodopsin from disk membranes in the dark without loss Of the absorption peak at nm, but when the solubilized protein is bleached by exposure to light, the ability to recover the original spectrum by rewith 1l-cis-retinal is usually lost, indicating an inability of the detergent to maintain opsin in a nativelike conformation. Available data for a variety of detergents with respect to this property were summarized in the preceding
'From the Whitehead Medical Research Institute and the Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710. Received September 19, 1980. Supported by National Institutes of Health Grant AM-04576. !part of this work was submitted in partial fulfillment of the requirements for the Ph.D. degree, Department of Biochemistry, Duke University. D.R.M. is presently a Dr. Chaim Weizmann Postdoctoral Fellow for Scientific Research in the Department of Physiology at Duke University.
0006-2960/81/0420-5212$01.25/0
0 1981 American Chemical Society
VOL. 20, N O .
AGGREGATION STATES OF RHODOPSIN
paper (Table I, McCaslin & Tanford, 1981), and experiments were described which demonstrate that recombination with recovery of the 500-nm absorption peak takes place when sodium cholate is used as a solubilizing detergent. In this paper, we compare the molecular properties of rhodopsin and opsin in sodium cholate with the corresponding properties in the nonionic detergent octaethylene glycol n-dodecyl ether (CI2Es),lin which recombination with recovery of the native spectrum has proved to be impossible. The most dramatic difference in the properties of the protein in the two detergent systems is the state of association, and therefore, a major part of this report is a comparison of the states of aggregation in the two detergents. Experimental Procedures
Materials. Dark-adapted bovine retinas were obtained from George A. Hormel & Co. (Austin, MN). Ficoll-400 and Con A-Sepharose were products of Pharmacia Fine Chemicals Inc. (Piscataway, NJ). C& was a chemically pure product produced by Nikko Chemicals Co. (Tokyo, Japan) and was used as received. Sodium cholate was obtained from Sigma Chemical Co. (St. Louis, MO) and was recrystallized several times from an acetonewater mixture after an initial treatment with activated charcoal followed by filtration through a Celite filter pad. Sodium cholate labeled in the carboxyl group with I4C (59.5 mCi/mmol) was obtained from Amersham Corp. (Arlington Heights, IL) and used without further purification. D20was obtained from Aldrich Chemical Co. (Metuchin, NJ). All other reagents were of standard laboratory grade. The Tes buffer referred to in this paper was 50 mM Tes, pH 7.5, and contained 100 mM NaCl unless otherwise indicated. Membrane Isolation. The procedure used was essentially that of Smith et al. (1975). The buffer was 0.1 M KH2P04 adjusted to pH 7.0 with NaOH and was saturated with argon. All membrane suspensions were kept at 4 "C under an argon atmosphere. In brief, 50 dark-adapted retinas were thawed and then suspended in sufficient 45% (w/v) sucrose in buffer to give a final volume of 75 mL. This suspension was forced through a disposable plastic syringe (without needle) and subsequently divided into two polyallomer centrifuge tubes. After centrifugation at 25 000 rpm for 1 h in a Beckman SW 27 rotor (Beckman Instruments, Palo Alto, CA), the crude rod outer segments were removed from the air-solution interface. These were again divided into two portions, diluted with buffer (final volume 50 mL in each tube), and pelleted. Each pellet was suspended in sufficient 40% (w/v) sucrose in buffer to fill a polyallomer tube, and the purified rod outer segments floated by centrifugation as described above. The rod outer segments were frozen or used immediately for the isolation of disk membranes. For the preparation of disk membranes, the purified rod outer segments were divided into two portions, diluted with argon-saturated water to 50 mL, and then pelleted. These pellets were suspended in 34 mL of 5% (w/v) Ficoll in argon-saturated water and allowed to sit for 1-3 h (longer incubation periods were found to decrease the final yield of membranes). The membrane suspensions in polyallomer centrifuge tubes were overlayed with 1.5 mL of water and centrifuged at 25 000 rpm in the Beckman S W 27 rotor for 2-2.5 h. The intact disks floating on the surface of the Ficoll Abbreviations used: CI2ES,octaethylene glycol n-dodecyl ether; Con A, concanavalin A; Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; NaDodS04, sodium dodecyl sulfate.
18,
1981
5213
solution were recovered by aspiration. The membranes were stored at -20 OC under argon in the 5% sucrose buffer. Protein Isolation. For the removal of sucrose prior to solubilization, the disk membranes were washed by centrifugation with Tes buffer which also contained 0.1 mM dithiothreitol. The disk membranes were suspended in a small amount of Tes buffer, and a concentrated detergent solution in Tes buffer was added until turbidity, as viewed through the infrared image converter, disappeared. For cholate, the final concentration was usually 40 mg/mL, and for CI2Es, the concentration used was approximately 10 mg/mL. After approximately 1 h, the solubilized sample was slowly run onto a small Con A-Sepharose column containing about 1.5 times more Con A sites than the total amount of rhodopsin being loaded onto the column. The column was washed extensively with detergent in Tes buffer (usually 20 mg/mL for cholate and 5 mg/mL for C12&) to remove proteins and lipids which were not specificially retained by Con A. The column was next washed with Tes buffer containing the final concentration of detergent needed for the experiment. The protein was eluted from the column matrix in a batchwise procedure using Tes buffer containing detergent and 0.5 M a-methyl mannoside. The eluted protein was dialyzed against buffer containing detergent to remove the mannoside. Spectroscopic Measurements. Absorption measurements were recorded at room temperature on a Cary 17D recording spectrophotometer equipped with a microcell adapter (Varian Instruments, Palo Alto, CA). The path length in all cases was 1.0 cm. The spectra were digitized by hand at 5-nm intervals, and these data were used for other manipulations, including replotting. The digitization of the spectra was adequate for most purposes; however, fine structure in the protein spectrum is lost. This fine structure consisted of typical protein spectral features including a tryptophan shoulder on the red side of the 278-nm maximum and a phenylalanine multiplet on the blue side. No attempt was made to reproduce these features in replotted spectra. Circular dichroic spectra were recorded at room temperature on a Jobin-Yvon Dichrographe I11 (J-Y Optical Co., Metuchin, NJ). Hand-digitized spectra were used for plotting and calculations. Since samples usually contained variable amounts of opsin, eq 1 was used to extract the molar ellipticity of
[el,
=
-
(l
-fR)[elOP
fR
(1)
rhodopsin, [e],, at each wavelength, where [eloBis the observed molar ellipticity for the unbleached sample based on total protein. [elop is the molar ellipticity observed after total bleaching (i.e., we assume that the opsin present initially in the sample has the same ellipticity as the newly formed opsin), and fR is the fraction of rhodopsin in the sample (see below). The mean residue weight estimated from published amino acid compositions was approximately 110. Sedimentation Studies. Both sedimentation velocity and equilibrium were performed in a Beckman Model E analytical ultracentrifuge equipped with a photoelectric scanner (Beckman Instruments, Palo Alto, CA). The distribution of protein in the centrifuge cell was recorded at 500 and/or 280 nm as dictated by the experiment at hand. To minimize bleaching when recording data at 500 nm, we changed the wavelength to 280 nm immediately after scanning past the meniscus of the solution column. When data were not being collected, the window into the vacuum chamber was blocked. Using this method, we have found that five to ten scans can be made
5214
BIOCHEMISTRY
MCCASLIN AND TANFORD
before there are detectable changes in Asoothrough the cell. To obtain the values of Mp(1 - 4’p) as a function of concentration, it was necessary to evaluate the slope of the equilibrium distribution at each point in the cell. For this purpose, a portion of the data (In A vs. 9)was fit by the method of least squares to a second-degree polynomial. The quality of the fit was assessed visually; slopes and absorbances were calculated in the region fitted by the curve. Another portion of the data overlapping the first set was fit in the same way, and this procedure was repeated until the entire set of data was smoothed (in some instances, an entire data set was adequately smoothed by a single quadratic equation). In regions of overlap, the slopes and absorbances obtained at the same value of 9 but using different smoothing functions were arithmetically averaged. The least-squares fits were performed by a Gauss-Jordan matrix inversion routine with full pivotal condensation (Dorn & McCracken, 1972). Plots of Mp(l - 4 ’ p ) as a function of absorbance were visually extrapolated to zero absorbance (i.e., protein concentration) to obtain the value of Mp(l - 4’p) for the minimum species. On the assumption that the curvature in the In A vs. 9plots is due to the presence of the oligomers of this smallest species, one can rigorously model the equilibrium distribution (Schecter et al., 1976). This modeling does not require any assumptions as to the mechanistic process giving rise to the oligomers. The model takes the form of a polynomial and is easily fit by least-squares methods. The sedimentation coefficients in C&8 were corrected to szo,wby assuming that the viscosity of the C&8 solutions at the concentrations employed was the same as that of water. For cholate at 20 mg/mL, the viscosity at 25 OC was 0.997 cP, and correction to szo,wwas made by assuming the temperature dependence to be the same as that of water. No correction for the effect of density on the sedimentation coefficient was included in the calculation of s20,w,but this will not significantly alter the reported values. Protein Assay. For spectral measurements and concentration determinations, 50 mM hydroxylamine was included in the sample to trap the retinal released by bleaching. For the estimation of rhodopsin content, we used 40600 M-’ cm-l for the extinction coefficient at 500 nm (Wald & Brown, 1953). Total protein was estimated by using 1.7 for the minimum value of the spectral ratio (AZE0/Am) and assuming that all was attributable to either rhodopsin or opsin. The fraction of rhodopsin CfR) in the sample is given by fR =
A500
1.7-
A280
The minimum value of A280/A500 is not absolutely established and may be expected to vary somewhat depending on the nature and the history of the preparation. There is, however, a general clustering of literature values near 1.7, and we have chosen to use this in our calculations. Furthermore, a value of 1.7 was the minimum found for rhodopsin preparations when using the vertical separation between the In A280 and In A s , data (in our centrifuge studies) as an estimate of A280/ ASoo.Values for total protein were occasionally determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard and found to agree with those calculated with eq 2. It is important to realize that none of the calculations in this paper depend on accurate knowledge of the protein concentration. Other Methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the methods of Weber & Osborn (1975) with modifications described by
Nielsen & Reynolds (1 978). The gels were stained for protein using Coomassie Brilliant Blue R-250(0.2% w/v) and diffusion destained. Samples were stained for carbohydrate using the periodic acid-Schiff base technique of Zacharius & Zell (1969). Retinal was covalently attached to the protein moiety by reduction of the Schiff base with NaBH, (1% NaBH, was added to the sample which was immediately exposed to light; 1% NaDodSO, was added and the sample subsequently dialyzed against the electrophoresis buffer). Fluorescent bands were observed visually using a hand-held ultraviolet light. Solution densities were measured with an Anton Paar DMA 02C densitometer (Mettler Instrument Co., Princeton, NJ). Viscosity measurements were made with Ostwald viscometers in a Neslab viscometer bath (Neslab Instruments, Inc., Portsmouth, NH). Phospholipid was assayed as total inorganic phosphate following wet ashing by the method of Bartlett (1959). Radioactivity was measured on a Beckman LS lOOC scintillation counter (Beckman Instruments, Palo Alto, CA). The counting cocktail was the dioxane-based scintillation fluid of Bray (1960). Curve fittings were performed on a Tektronix 3 1 calculator equipped with a Tektronix 466 1 digital plotter (Tektronix, Beaverton, OR)using programs written by one of us (D.R.M.). All procedures involving rhodopsin were performed by using infrared illumination (flashlight with infrared filter from Edmund Scientific Co., Barrington, NJ; less than 5% T for X 0.9 cm3/g, the ODdependent term can be blanked out altogether at a value of p that is experimentally accessible by varying the D 2 0 / H 2 0 ratio. Because of the small difference between 8, and OD in the present experiments, however, the method is not able to provide a significant discrimination between the possibilities permitted by the data at one solvent density alone, as the following results show. We substituted D 2 0 for H20by repeated dialysis against a buffer solution prepared with pure D20. Sedimentation equilibrium experiments and data analysis were carried out as before, but different d values were used because both the protein and the detergent contain exchangeable protons which will affect their mass but will have little effect on the partial molar volume (Edelstein & Schachman, 1967). Rhodopsin contains a maximum of about 480 exchangeable protons per polypeptide chain, but not all of these were observed to exchange in a 36-40-h period in hydrogen-isotope exchange experiments (Osborne & Nabedryk-Viala, 1978). Using 300
V O L . 20, N O . 18, 1 9 8 1
AGGREGATION STATES OF RHODOPSIN
- 100
0 100 200 Detergent Binding (mols/rnonomer)
- 40
0 50 IO0 Detergent Binding (mols/monomer)
300
n I
0 X
FIGURE 5: Calculated values of M and 6D required to give the experimentally observed values of Mp(f - d’p). The upper panel shows the calculations for CI2E8in Tes buffer using M (1 - d’p) = 12 300 and p = 1.01 g/mL; the other constants neede8for eq 3 are in the text. The lower panel presents the same calculations for rhodopsin in cholate with Tes buffer in H20(p = 1.01 g/mL) and Tes buffer in D20(p = 1.11 g/mL). In these calculations, the partial specific volumes are corrected for deuterium incorporation as discussed in the text. Maximum binding refers to the limit set by a dialysis equilibrium experiment described in the text. Monomer, dimer, trimer, and tetramer refer to oligomers of the opsin polypeptide chain (assuming a molecular weight of 38 500). Mp(1 - 4’p) in water is 33 OOO f 2000 and 24000 f 2000 in D20.The two vertical bars represent the limits to the theoretical curves calculated by using the maximum errors of the value for Mp(1 - d’p) in water.
for the number of actually exchangeable protons leads to an increase in the polypeptide chain molecular weight of 0.78%, which is of course negligible since we are trying to discriminate between the molecular weights for trimeric and dimeric rhodopsin. However, the change of D, (from 0.735 to 0.728 cmn3/g) is significant because it is the change in 1 - D@ that affects the difference in the results between the two solvents. Cholate ions have three exchangeable protons, and OD is expected to change as a result from 0.771 to 0.776 cm3/g. The expected effect on aD is negligibly small. The limiting value of M,(1 - 4’p) obtained in D 2 0 was 24 000, as compared to 33 000 in HzO.The mutually compatible values of Mpand aD for both sets of data are shown in Figure 5. Imposing the condition that both Mpand aD must have the same values in the two solvents, we see that the results favor a molecular weight corresponding to a trimer of the polypeptide chain, with aD approximately 0.1-0.2 g/g, corresponding to about 10 or 15 bound cholate ions per polypeptide chain or 30-50 bound ions per trimer. The significance of this result depends on the experimental uncertainty in the limiting values of Mp(1 - 4’p) in the two solvents. Because the numbers were obtained by visual extrapolation, a rigorous statistical analysis becomes difficult and was not used. Most of the extrapolations gave limiting values in the range of 33 000 f 1500 (the experimental error for a single determination in the ultracentrifuge is less than 5%), and none of the values were outside the range 33 000 f 2000. Even a 5% uncertainty in Mp(l - 4’p) is, however, sufficient to put the difference between the two curves of Figure 5 at Mp = 77000 (corresponding to a dimer of the polypeptide chain) within exper-
5217
imental error. The data thus show that the results in D 2 0 are consistent with those in HzO, but the extent to which they favor the trimer value of Mp over the dimer value must be taken as slight. A better discriminant between the two possible values of Mpis provided by the fact that the amount of detergent that binds to a solubilized membrane protein is normally related to the size of the micelle that the detergent forms in the absence of protein. Both cholate and deoxycholate form very small micelles (Small, 1971) and bind to membrane proteins in lower molar ratios than do other detergents. There are no previously reported measurements for cholate, but measurements for deoxycholate have been made for cytochrome bS (Robinson & Tanford, 1975) and the sarcoplasmic reticulum CaATPase (LeMaire et al., 1975), under conditions where both are monomeric, and for the dimeric coat protein of bacteriophage (Makino et al., 1975). All values fall within the range of 25-40 mol of bile salt bound per mol of solubilized particle. The binding required if rhodopsin is a trimer, as given above, is within this range. As Figure 5 shows, an Mpvalue of 77 000, corresponding to the dimeric association state, requires a binding of about 0.75 g/g, corresponding to 135 mol of cholate per solubilized particle which is outside this range. While present data do not allow an unequivocal choice between dimer and trimer as the association state, we will for the purposes of this discussion refer to the cholate particle as a trimer of rhodopsin. State of Association in C,,E,. Sedimentation equilibrium experiments in C&8 were made by using rhodopsin samples whose opsin content was similar to that of the samples used for the data obtained in sodium cholate (i.e,, 47 500. Thus, rhodopsin in C12E8 is monomeric. Allowing an uncertainty of f2000 in the polypeptide chain molecular weight (nominally 38500) leads to a aD value of 3.5 f 1 g/g or 250 f 60 mol/mol. Sedimentation velocity measurements in C& were made, using the absorbance at 500 nm to measure the boundary movement arising from unbleached rhodopsin. The data were consistent with sedimentation of a single species with an uncorrected sedimentation coefficient at 21.5 “C of 2.26 S. The corresponding value of s20,w is 2.13 S. Effect of Bleaching. The sedimentation equilibrium experiments described above show that opsin present in retinas shipped to us exists in the same state of association as that of unbleached rhodopsin after solubilization in sodium cholate, but not after solubilization in C12E8. A similar result was obtained when rhodopsin was partially bleached by exposure
5218
B IO C H EM IST R Y
MCCASLIN A N D TANFORD
r 2 icrn')
Effects of light on aggregation state of rhodopsin in CI2&. The sample was initially 6.2 ~ I Mrhodopsin in Tes buffer (the concentration of Tes was 20 mM in this experiment, but otherwise the buffer was as described in the text) containing 1 mg/mL CI2E8,and A280/ASW = 2.3, initially. The speed was 20000 rpm, and the temperature was 18.97 "C. Only data collected at 280 nm are shown, and the symbols are as described in Figure 6. FIGURE 7:
I
47.5
I
I
I
1
I
I
48.5
49.5
50.5
l
i 51.5
r 2 (crn') FIGURE 6: Effects of light on aggregation state of rhodopsin in cholate. The samples initially contained 7.5 ~ I Mrhodopsin in Tes buffer containing 20 mg/mL cholate, Azso/Am = 2.6 initially. The speed was 9000 rpm, and the temperature was 20.02 OC. The details of the experiment are discussed in the text. (0)Sedimentation equilibrium gradient established prior to bleaching; (0)distribution of absorbing materials immediately following in situ bleaching; (0) distribution 24 h after in situ bleaching; (m) distribution at equilibrium after remixing sample.
to light in the untracentrifuge after the equilibrium distribution for the unbleached sample had been established. The results in sodium cholate are shown in Figure 6. About 20 min were required to obtain a decrease of about 40% in Asooat one point in the cell, and the whole cell was scanned immediately after that. Provided no significant redistribution of the protein occurs during the bleaching process, this initial scan is expected to show a decrease in ASwrbut the plot of In Am vs. 9 should remain parallel to that of the original scan. The data showed the expected behavior, except at the very lowest absorbance values near the meniscus of the solution column. A similar deviation from the theoretical result was obtained in C&g, and it is probably due to small differences in the incident light intensity falling on the sample in different parts of the cell, so that the extent of bleaching is not quite the same everywhere in the cell. The sample was then allowed to spin in the ultracentrifuge for a sufficient time to allow reequilibration to occur (28 h) and was then scanned again. No significant change in the data obtained at 500 nm was expected, because the data at this wavelength monitor the distribution of the remaining unbleached rhodopsin, and as Figure 6 shows, no change was observed. As a final check, the run was terminated, the sample was stirred in the dark, and a new equilibrium was carried out. Again no change was observed, except that the anomalous low absorbance near the meniscus of the cell disappeared. The important part of this experiment lies in the results at 280 nm, where the freshly bleached opsin will make a major
contribution. As the lower panel of Figure 6 shows, all four scans at this wavelength were indistinguishable. Bleaching clearly has no effect on the association state in sodium cholate, and since even the smallest species in this detergent is a trimer, it is likely that many of the oligomeric complexes will be hybrids containing both unbleached and bleached molecules. Quite different results were obtained when this experiment was repeated with rhodopsin in C&g. Figure 7 shows the results obtained at 280 nm. A continuous loss of absorbance is observed, reflecting the formation of high aggregates that move rapidly to the bottom of the cell, and formation of intermediate oligomers is reflected in curvature of the plots as the bottom of the cell is approached. The portion of the plots near the meniscus remained linear, with a slope corresponding to the monomer molecular weight, as observed in the original sample. It is of interest to note that the dramatic difference between the effect of bleaching on rhodopsin in sodium cholate and in C&, with respect to both the state of association and the ability to recombine with 1 1-cis-retinal, is not accompanied by a major difference in the secondary structure of the protein. Figure 8 shows that the effect of bleaching on the circular dichroic spectrum of the protein is nearly the same in the two detergents. If one analyzes the results in terms of empirical procedures such as described as by Greenfield & Fasman (1969), complete bleaching gives rise to a 6-8% decrease in a-helical content when in sodium cholate and a 6-9% decrease when in C&g. Experiments with partially bleached samples (not shown) demonstrated that the change in ellipticity seen in Figure 8 is proportional to the extent of bleaching. In Figure 1, the spectrum of bleached rhodopsin in ClzEs is seen to have a tailing absorption for wavelengths greater than 400 nm. Our sedimentation studies have clearly shown that opsin in C& aggregates extensively; hence, we attribute the aforementioned absorption tail to light scattering arising from aggregated opsin. Association States in Sodium Cholate and CIzEBAre Equilibrium States. In order to determine whether the tendency of rhodopsin to associate to an oligomeric state with a defined minimal molecular weight is an inherent thermodynamic property of the molecule, reversibility of the dissociation to monomers in C1& was tested. Disk membranes were solubilized in CI&, and the rhodopsin was bound (in the dark) to a Con A column. Since purified rhodopsin is monomeric at the detergent concentration employed, it is
VOL. 20, NO. 18, 1981
AGGREGATION S T A T E S O F RHODOPSIN
5.0
5219
in sodium cholate, with a well-defined minimal molecular weight corresponding to a trimer of the polypeptide chain. The simplest interpretation of this result in relation to the loss of reversibility upon bleaching in C12& is that the oligomeric state is representative of the native state of rhodopsin in the disk membrane. Consistent with this conclusion, the data from freeze-fracture electron microscopy (Chen & Hubbell, 1973; Corless et al., 1976) suggest that the intramembranous particles in the plane of the disk membranes could contain about four monomeric rhodopsin molecules. X-ray diffraction data (Blaurock, 1977) and measurements of rotational diffusion time (Cone, 1972; Baroin et al., 1978) have, however, been interpreted in terms of a monomeric membrane-incorporated molecule. It is perhaps of interest in this connection that the native state of bacteriorhodopsin in the crystalline purple membrane of Halobacter halobium consists of an array of trimeric protein molecules (Henderson & Unwin, 1975), and there is evidence (Rehorek & Heyn, 1979) for a relation between trimer formation and retinal binding (in this case, trans-retinal). However, a trimer appears not to be required for the biological function of the protein as a light-driven proton pump (Dencher & Heyn, 1979). If native rhodopsin is in fact a trimer, it is important to note that the dissociation of the unbleached protein to a monomeric state is of itself not an irreversible process, as demonstrated by the reversibility of the dissociation described above. The effect of the association state is manifested only after bleaching has occurred, suggesting that protein-protein interactions in the oligomeric state (in sodium cholate) are sufficient to retain some feature of the protein conformation required for recombination with 11-cis-retinal, whereas this feature is lost after the monomer is formed (at least in C12E8). It is important to mention in this connection that Hubbard (1954) purported to show that rhodopsin is monomeric in digitonin, another detergent in which the ability to recombine with 11-cis-retinal is preserved after bleaching. The validity of her conclusion is however questionable: Hubbard measured the molecular weight of the digitonin-solubilized particle as 260000, with a measured partial specific volume of 0.766 cm3/g. She then assumed that all of the digitonin in the extracting buffer was bound to the protein, which led to a aD value of more than 5 g of digitonin per g of protein, and on this basis concluded that each particle contained only one rhodopsin molecule. In the absence of direct binding measurements, it is impossible to estimate a true value for aD, but it should be noted that digitonin contains a fused ring system similar to that of cholate and that a aD value closer to the value we observed for cholate (-0.2 g/g) might therefore have been expectede2 The sedimentation velocity measurements reported in this paper allow one to make an estimate of the asymmetry of the sedimenting particle (Tanford et al., 1974). The limiting value at high cholate concentration (Figure 2) is 4.7 S under the conditions used, which corresponds to szo,w= 4.57 S, or to a Stokes radius (Rs)of 59 A. The minimal radius of a dry particle containing a trimer of rhodopsin with dD = 0.2 g/g
L
1
200
I
I
1
215
230
245
Wavelength (nrn)
FIGURE8: Circular dichroic spectra of rhodopsin in cholate and C12E&.
The solid lines represent the spectra of unbleached rhodopsin extracted, as described in the text, from the recorded spectrum of the rhodopsin-opsin mixture. The dashed lines are the totally bleached samples. The CL2E8sample was 2.9 p M rhodopsin (A280/ASM) = 2.4) in Tes buffer (Tes concentration was 20 mM) containing 0.1 mM dithiothreitol and 1 mg/mL C12E8; the path length of the cell was 0.1 cm. = 2.6) in the The cholate sample was 14 p M rhodopsin (A280/ASM) standard Tes buffer containing 20 mg/mL cholate; the path length was 0.02 cm.
presumably bound to the column as a monomer. The column was washed with to remove phospholipid and then with cholate at a concentration of 20 mg/mL. At this point, one presumably has monomeric rhodopsin with cholate bound to it, attached to the column through its carbohydrate moiety. The rhodopsin was then eluted from the column and subjected to sedimentation equilibrium. It was found to behave identically with rhodopsin that had never been exposed to C1&, with respect to both the state of association of the originally eluted sample (Figure 3) and the effect of bleaching on it (Figure 6). The monomer and trimer states of the protein thus clearly reflect a reversible thermodynamic equilibrium. Moreover, this experiment adds further support to our earlier contention that the oligomers in cholate are not the result of disulfide bond formation since the oligomeric state is induced by merely exchanging Cl& for cholate.
~~
Discussion
This paper has compared the properties of rhodopsin in esstentially lipid-free form in two detergents. In one of the detergents (sodium cholate), the native state of the protein after bleaching can be recovered by recombination with 11cis-retinal; in the other (CIZE8), bleaching is irreversible. We have found that the protein differs in its state of aggregation in the two detergents, being monomeric in Cl& but oligomeric
~~
~
~
* The possibility of lipid retention in the solubilized particle was also
ignored. Since rhodopsin (0 = 0.735 cm3/g) and digitonin (0 = 0.738 cm3/g) have nearly the same partial specific volume, the observed B of 0.766 cm3/g in fact requires the presence of phospholipid, to the extent of about 12%, using a reasonable value for the 0 of the lipid. [The presence of lipid in the particle has been demonstrated experimentally by Krinsky (1958).] The combined mass of protein and digitonin in the Hubbard particle is thus 229000 rather than 260000, but one still needs to reduce bD to no lower than 1 g/g to obtain a protein mass corresponding to the trimeric association state.
5220 B I 0 C H E M I S T R Y is 34 A, so that Rs/Rmi, [which is identical with f/fminas defined by Tanford (1961)l has a value of 1.72. Though both hydration and asymmetry contribute to this parameter, hydration alone cannot conceivably account for the large value of Rs/R,,,,,, observed (it would require more than 4 g of bound water per g of protein), and the result obtained thus indicates that the rhodopsin-cholate particle must by highly asymmetric. Because the sedimenting particle is trimeric, the result cannot yield a quantitative measure of the asymmetry of an individual rhodopsin molecule, but the conclusion is in qualitative agreement with other data that indicate rhodopsin to be a highly asymmetric molecule (Wu & Stryer, 1972; Sardet et al., 1976; Osborne et al., 1978; Wright, 1976; Stryer, 1978). = The sedimentation coefficient measured in CI2E8 2.1 3 S) leads to the much smaller value Rs/R- = 1.24, which suggests that the sedimenting particle in this case is fairly symmetric. Because of the high detergent content of the particle (6, 3.5 g/g), it is not possible to draw any conclusions about the asymmetry of the rhodopsin molecule within the particle. Other physical and chemical data, not related to the state of association, suggest that rhodopsin solubilized in cholate has a conformation resembling the conformation of membrane-bound rhodopsin and differing from the conformation of rhodopsin solubilized in detergents where the recombination capacity is lost. For example, Fung & Hubbell (1978) reported that the proteolytic fragments produced from cholate-solubilized rhodopsin were identical with those from membrane-bound rhodopsin. Furthermore, they demonstrated that the proteolytic fragments of cholate-solubilized rhodopsin did not dissociate upon photolysis as is the case for rhodopsin solubilized in detergents in which the recombination reaction has been lost (Fung & Hubbell, 1978; Pober & Stryer, 1975). Since proteolytically cleaved membrane-bound rhodopsin is capable of the recombination reaction (Pober & Stryer, 1975) and since there is no effect of light on the chemical crosslinking behavior of the proteolyzed rhodopsin (Fung & Hubbell, 1978), it is probable that the proteolytic fragments remain associated in the membrane whether bleached or unbleached. The central role of rhodopsin in visual transduction and its implied interactions with other components (e.g., enzymes) in the rod outer segments provide a great impetus to find a detergent system in which all the properties attributable to native membrane-bound rhodopsin are preserved. As indicated in the introduction, rhodopsin can be isolated in nearly all detergents without loss of its native absorption spectrum, but in most of these detergents, the ability of opsin to recombine with 1 l-cis-retinal is lost. We have begun to systematically examine rhodopsin in various detergents in an attempt to elucidate the molecular properties involved in maintaining the ability of opsin to undergo recombination. For rhodopsin in ClzEs and various other detergents in which recombination is lost, there are data demonstrating or at least suggesting that the unbleached protein is monomeric and undergoes aggregation upon photolysis. Clearly, in these systems, any alteration in a physical or chemical property which occurs upon bleaching should not be interpreted on an a priori basis as playing a role in the visual process. We have found that in sodium cholate (in which the recombination capacity is intact) the protein is oligomeric and that this association is not affected by bleaching. While the present data base is too small to prove that association is essential to the maintenance of the recombination capacity of rhodopsin, the strong correlation between the loss of recom-
-
MCCASLIN AND TANFORD
bination and a monomeric rhodopsin in a variety of detergents is very suggestive. Acknowledgments We are grateful to Hoffmann-La Roche Inc. for the gift of 1 l-cis-retinal. We are grateful to Drs. J. M. Corless and J. A. Reynolds for many helpful and critical discussions. References Albert, A. D., & Litman, B. J. (1978) Biochemistry 17, 3893-3900. Baroin, A., Thomas, D. D., Osborne, D., & Devaux, P. F. (1978) Biochem. Biophys. Res. Commun. 78, 442-447. Bartlett, G. R. (1959) J . Biol. Chem. 234, 446-468. Blaurock, A. E. (1977) in Vertebrate Photoreception (Barlow, H. B., & Fatts, P., Eds.) pp 61-76, Academic Press, London. Bray, G. A. (1960) Anal. Biochem. I, 279-285. Chen, Y. S., & Hubbell, W. L. (1973) Exp. Eye Res. 17, 517-532. Cone, R. A. (1972) Nature (London),New Biol. 236, 39-43. Corless, J. M., Cobbs, N. H., 111, Costello, J. M., & Robertson, J. D. (1976) Exp. Eye Res. 23, 295-324. Daeman, F. J. M., De Grip, W. J., & Jansen, P. A. A. (1972) Biochim. Biophys. Acta 271, 419-428. Dencher, N. A., & Heyn, M. P. (1979) FEBS Lett. 108, 307-3 10. Dorn, W. S., & McCraken, D. D. (1972) Numerical Methods with Fortran ZV Case Studies, Wiley, New York. Edelstein, S . J., & Schachman, H. K. (1967) J . Biol. Chem. 242, 306-3 1 1. Fung, B. K.-K., & Hubbell, W. L. (1978) Biochemistry 17, 4396-4402. Greenfield, N., & Fasman, G. D. (1969) Biochemistry 8, 4108-4116. Henderson, R., & Unwin, P. N. T. (1975) Nature (London) 257, 28-32. Henselman, R. A., & Cusanovitch, M. A. (1974) Biochemistry 13, 5199-5203. Hubbard, R. (1954) J . Gen. Physiol. 37, 381-399. Hubbell, W. L., & Bownds, M. D. (1979) Annu. Rev. Neurosci. 2, 17-34. Krinsky, N. I. (1958) Arch. Ophthalmol. (Chicago) 60, 688-694. LeMaire, M., Jorgensen, K. E., Roigaard-Petersen, H., & Moller, J. V. (1976) Biochemistry 15, 5805-5812. Lewis, M. S., Kreig, L. C., & Kirk, W. D. (1974) Exp. Eye Res. 18, 29-40. Lowry, 0 .H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. Makino, S., Woolford, J. L., Tanford, C., & Webster, R. E. (1975) J . Biol. Chem. 250, 4327-4332. McCaslin, D. R. (1979) Ph.D. Dissertation, Duke University. McCaslin, D. R., & Tanford, C . (1981) Biochemistry (preceding paper in this issue). Nielsen, T. B., & Reynolds, J. A. (1978) Methods Enzymol. 48, 3-10. Osborne, H. B., & Nabedryk-Viala, E. (1978) Eur. J . Biochem. 89, 81-88. Osborne, H. B., Sardet, C., Michel-Villax, M., & Chabre, M. (1978) J . Mol. Biol. 123, 177-206. Plantner, J. L., & Kean, E. L. (1976) J . Biol. Chem. 251, 1548-1 552.
Biochemistry 1981,20, 5221-5226 Pober, J. S., & Stryer, L. (1975) J. Mol. Biol. 95,477-481. Rehorek, M., & Heyn, M. P. (1979) Biochemistry 18,
4977-498 3. Reynolds, J. A., & Tanford, C. (1976)Proc. Natl. Acad. Sci. U.S.A. 73, 4467-4477. Robinson, N. C., & Tanford, C. (1975) Biochemistry 14,
369-378. Sale, G. J., Towner, P., & Aktar, M. (1977)Biochemistry 16,
5641-5649. Sardet, C., Tardieu, A., & Luzzati, V. (1976)J. Mol. Biol. 105, 383-407. Schechter, N. M., Sharp, M., Reynolds, J. A,, & Tanford, C. (1976)Biochemistry 15, 1897-1904. Small, D. M. (1971) in The Bile Acids (Nair, P. P., & Kritchevsky, D., Eds.) Vol. 1, pp 249-353, Plenum Press, New York. Smith, H. G., Jr., Stubbs, G. W., & Litman, B. J. (1975)Exp. Eye Res. 20, 21 1-217.
5221
Stryer, L. (1978) Annu. Rev. Biochem. 47, 819-846. Suzuki, T., Makino, M., & Azuma, M. (1977) Biochim. Biophys. Acta 494, 115-125. Tanford, C.(1 961) Physical Chemistry of Macromolecules, Wiley, New York. Tanford, C., Nozaki, Y., Reynolds, J. A., & Makino, S. (1974) Biochemistry 13, 2369-2376. Tanford, C.,Nozaki, Y., & Rhode, M. F. (1977) J. Phys. Chem. 81, 1555-1 560. Wald, G., & Brown, P. K. (1953) J . Gen. Physiol. 37,
189-200. Weber, K., & Osborn, M. (1975) Proteins, 3rd Ed. 1,
179-223. Wright, A. K. (1976)Biophys. Chem. 4, 199-202. Wu, C.-W., & Stryer, L. (1972)Proc. Natl. Acad. Sci. U.S.A. 69, 1104-1 108. Zacharius, R. M., & Zell, T. E. (1969)Anal. Biochem. 30,
148-152.
Comparison of Binding Sites for Wheat Germ Agglutinin on Raji Lymphoblastoid Cells and Their Isolated Nuclei and Plasma Membranes? Marti Jett* and G. A. Jamieson ABSTRACT:
Raji lymphoblastoid cells and the cell nuclei and plasma membranes isolated by the glycerol-lysis technique [Jett, M., Seed, T., & Jamieson, G. A. (1977)J. Biol. Chem. 252,2134-21421 have been examined for their ability to bind wheat germ agglutinin. Intact cells and isolated nuclei showed similarities (i) in the total number of binding sites (3.38 X lo6 and 4.06 X lo8,respectively), indicating at least a 2-fold higher receptor density on the nuclei, (ii) in the ratios of the number of high-affinity sites and low-affinity sites (1.05and 1.07),and (iii) in the apparent association constants at the high-affinity
sites (28 nM and 48 nM) and at the low-affinity sites (116 nM and 370 nM). Isolated plasma membranes had a similar number of total binding sites calculated on an equivalent cell basis (2.01 X lo6)but showed differences in the ratio of highto low-affinity sites (1.5) and in their apparent association constants (3 nM and 22 nM). These results suggest similarities in the lectin receptors on the outer surface of lymphoblastoid cells and the cell nuclei. The differences obtained with isolated membranes may be due to inversion of the membrane vesicles or to their decreased rigidity as compared with the intact cell.
T e relationship between the plasma and nuclear membranes of cells is not clear but is of obvious importance in understanding membrane biosynthesis, cell growth, and neoplastic transformation. Morphologically, the two membrane systems may be continuous (Grundmann, 1699), and they are known to share enzymes such as 5’-nucleotidase (Crumpton & Snary, 1974;Wallach & Winzler, 1974) and thymidine-5’-phosphodiesterase (Erecinska et al., 1969;Jett et al., 1977). Latins have been used extensively to elucidate relationships between the cellular membrane comparments. Fluorescent and ferritin-conjugated lectins such as concanavalin A‘ and wheat germ agglutinin, which bind to intact cells, also bind to bovine liver nuclei (Nicolson et al., 1972) and to rat liver nuclei (Virtanen & Wartiovaara, 1976). Internalization of Con A has been observed with bovine hepatic cells under normal incubation conditions within 1-2 h (Nicolson, 1974). Ricinus communis lectin and phytohemagglutinin have been shown to interalize, presumably while bound to their surface receptors, into the GERL of mouse ganglion neurons within 1-3 h upon incubation at 37 OC (Gonatas et al., 1977). The
ultimate fate of the internalized lectin is not known in either case. However, quantitative comparisons of the distribution and nature of the lectin binding sites on cells and nuclei are lacking. WGA binds to surface glycoproteins in a number of different cell types (Nagata & Burger, 1974;Goldstein & Hayes, 1978), and the lectin specifically is directed mainly toward GlcNAc, although AcNeu is about one-half as effective (Peters et al., 1979; Bhavandan & Katlic, 1979). We have now quantitated the binding of wheat germ agglutinin to high- and low-affinity sites on intact Raji lymphoblastoid cells and their isolated nuclei and plasma membranes prepared by this procedure. A preliminary account of some aspects of this work has already appeared (Jett, 1977).
t From the American Red Cross Blood Services Laboratories, Bethesda, Maryland 20014. Received January 14, 1981. This investigation was supported, in part, by US.Public Health Service Grants HL 20971, HL 14697, and RR05737. Contribution No. 452 from the American Red Cross.
Materials and Methods Sepharose-bound WGA was obtained from Vector Laboratories (Burlingame, CA). [3H]Glucosamine (1 73 mCi/mg, uniformly labeled) was obtained from Amersham/Searle (Arlington Heights, IL). I Abbreviations used: Con A, concanavalin A; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline; GlcNAc, N-acetylglucosamine; C13CCOOH,trichloroacetic acid; AcNeu, N-acetylneuraminic acid.
0006-2960/81/0420-5221$01.25/0 0 198 1 American Chemical Society
